Virus filtration of cell culture media

ABSTRACT

The invention relates to a method for removing a viral contaminant from a preparation, being a cell culture medium or at least a component of a cell culture medium. The method comprises subjecting said preparation to filtration for at least about 24 hours through a virus filter having an effective pore size of maximum about 75 nm. Further, the invention relates to the use of a virus filter in filtration of at least about 24 hours, wherein the virus filter has an effective pore size of maximum about 75 nm for the removal of viral contaminant from a preparation, being a cell culture medium or at least a component of a cell culture medium. In some embodiments the filtration according to the invention operates at a volumetric capacity of at least about 2000 L/m 2 . Further, the invention relates to the use of a preparation, being a cell culture medium or at least a component of a cell culture medium obtainable according to method of the invention for cell culture; pharmaceutical, diagnostic and/or cosmetic preparations as well as in food preparations.

The present application is a divisional of U.S. application Ser. No.13/921,417, filed Jun. 19, 2013, which claims priority from U.S.Provisional Application Ser. No. 61/662,814, filed Jun. 21, 2012. Theentire contents of those applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Virological safety is a significant concern in the biopharmaceuticalindustry. Despite efforts to mitigate the risk, incidents involvinglarge-scale viral contamination of biologics have raised concern in theindustry. Highly profiled events include, for example, Genzyme's 2009detection of a Vesivirus 2117 contamination of its CHO (Chinese hamsterovary) cell culture which halted production of Cerezyme® and Fabrazyme®and Merck's 2010 contamination of its Rotarix® vaccine by porcinecircovirus 1. A likely source of contamination is at the cell culturestage. In addition to the economic toll on the manufacturing company(one report puts the estimate at over hundred million loss per 10000 Lbioreactor contamination plus fines from the agencies), such events posea risk to patients and disrupt access to the biopharmaceutical products(Liu et al., Biotechnol. Prog. 2000, 16, 425-434). As a result, there isheightened regulatory scrutiny and demand for new techniques to detect,prevent, and remediate viral contaminations.

In general, viral contaminants can be differentiated into upstream anddownstream viral contaminations. Downstream contaminations may becontrolled by applying closed systems, however, especially upstreamcontaminations are difficult to control and detect even by extensivetesting. Viral contaminants may also originate from the use of animalderived materials in the biopharmaceutical production. Where theproduction cell line is free of extraneous viral contaminants andproduction does not involve use of animal derived materials, viralcontaminants could still enter by way of cell culture media. Forinstance, synthetic media may be supplemented with recombinant growthfactors produced in a serum-supplemented system and protein-free mediummay nevertheless contain filtered protein hydrolysates. However, viralcontamination may even occur in completely chemically defined medium,because large quantities of medium components may be packed innon-sterile containers. Conventional sterilizing-grade filters areneither designed to nor capable of safeguarding against viralcontaminants, so other measures must be employed to ensure virologicalsafety.

Detection of adventitious viruses at one or more checkpoints of theproduction process is standard practice. However, detection alone is aninadequate measure against viral contamination of biopharmaceuticalproducts, especially where the viral contaminant present is unsuspected,unknown, or an emerging viral agent. Such viral agents can escapedetection by even well-designed DNA microarrays representative of alarge collection of sequenced viruses. The challenge is furthercompounded by the low levels of viral contaminants needed to infect acell culture and currently limited detection assay sensitivity.

High titers of the viral contaminant may not manifest in the form ofaltered cell culture parameters, e.g. culture density, protein titers,beyond their normal range. On the other hand, infectivity assays arehighly specific and require different conditions for each virus. As aresult of viral contamination, downstream equipment, fluids, andproducts can be tainted, incurring millions of dollars in batch setup,waste disposal, lost sales, and decontamination. Thorough screening ofraw materials for viruses is difficult due to sample heterogeneity andthe large volumes involved in biopharmaceutical production processes.

Viral clearance techniques can be classified into one of two groups:inactivation and filtration. Inactivation methods seek the irreversibleloss of viral infectivity, whereas filtration methods seek tomechanically reduce the viral contaminant. Conventional inactivationmethods employ ultraviolet (UV) irradiation, gamma irradiation, heat,low or high pH, or solvent/detergent exposure. In instances where UVirradiation can effectively and irreversibly eliminate viral activity,it may be impractical on a large-scale basis or unsuitable for preparedmedia. Autoclaving, while possible for heat-stable liquids, may altersensitive media. An alternative method known as high-temperature,short-time (HTST) heat treatment is not as harsh but demands costlyequipment, automation, and validation procedures to preserve the mediacharacteristics. Low or high pH exposure is ineffective across thespectrum of possible viral contaminants and can negatively impact thequality or osmolarity of the media. Solvent/detergent exposure islikewise not a broad-spectrum solution and is effective only for viruseswith a lipid envelope. As such, the ideal method should balance costconsiderations and the needs to effect viral clearance in raw materialsand provide a broad-spectrum solution without compromising growth rateor yield.

Viral-retentive filtration offers the appropriate balance. It does notchemically alter media components and is suitable for use withheat-sensitive feed/media. Furthermore, viral-retentive filtration is abroad-spectrum solution since it operates on a size exclusion principle.However, viral-retentive membranes are costly (approximately about 2000to 5000 EUR per m²). The low specific flow rates characteristic offiltration of media volumes have made the method economically taxing ona scale suitable for large scale bioreactor supply, due to the cost ofthe membrane area needed. For example, where virus filtration isconnected in-series to sterilizing grade media filtration, virusfiltration preferably needs to occur within a working day, i.e. amaximum of 2 to 10 hours after preparation of the bulk medium in orderto prevent contamination of the bulk medium. Therefore, a largefiltration area is needed to stay within this critical time window,which in turn raises costs.

Surprisingly, it has been found that the drawbacks of said prior artvirus filtration can be overcome by filtration of the respectivepreparation, being a cell culture medium or at least a component of acell culture medium, for at least about 24 hours through a virus filterhaving an effective pore size of maximum 75 nm. If the required volumeof the respective preparations is filtered during a longer time frame,i.e. for at least 24 hours the volumetric capacity of the virus filtersincrease enormously. Surprisingly, it has been found additionally thatsignificant overall virus titer reduction can be achieved over thisextended period of time. This is especially beneficial in upstream virusremoval in cell culture systems.

Therefore, the method of the invention enhances the economic efficiencyof virus filtration by enhancing throughput and volumetric capacity,respectively. The method according to the present invention operates ata volumetric capacity of at least 2000 L/m², thereby helping to maximizethe use of high capacity virus filters, decreasing the effective costsassociated therewith, and presenting a solution practicable on a largescale and readily integrable into existing production processes.

The enormous impact of the method according to the invention and theinventive use of the respective virus filters on sterile manufacturingprocesses, in particular processes where sterile preparations, e.g. cellculture media and buffers, are used can be understood by means of thefollowing example. Assuming that a square meter of a virus filtermembrane costs about 3000 EUR in average and a cell culture medium isused costing about 10 EUR per liter medium, then the costs for 1000 Lvirus filtered media are 13 EUR per liter medium, which increases thecosts of goods for media preparation by about 30%. If 2000 L can befiltered with a virus filter membrane then the costs decrease to 11.50EUR. Further increase of volumetric capacities, e.g. beyond 5000 Lreduces the costs to less than 0.6 EUR per liter medium, which makes theadditional costs for providing a virus filtered medium considerably low.As a result, the high costs of using virus filters, in particular inupstream decontamination of potential viral or viral contaminationdecreases significantly by increasing the volumetric capacity of thevirus filtration method.

The present invention fully addresses this problem of high costs and lowvolumetric capacity of virus filters, respectively. The volumetriccapacity of the virus filter can be increased by performing the virusfiltration for at least about 24 hours through a virus filter having aneffective pore size of maximum 75 nm. Surprisingly, it has been foundthat the volumetric capacity of the used costly virus filters can bebetter exploited leading to a 2 to 100-fold increase of the volumetriccapacity while maintaining the filter integrity. Although a 2 to 3-foldincrease of volumetric capacity already has a great impact to theproduction process and the related production costs, with the methodaccording to the invention an up to 100-fold increase of volumetriccapacity or even more can be achieved. This offers great opportunitiesand makes viral removal cost efficient even with costly virus filtersthat now can be used to further improve viral safety in cell cultureprocesses, in particular in upstream viral removal of cell cultureprocesses, pharmaceutical, diagnostic and/or cosmetic and foodprocesses.

SUMMARY OF THE INVENTION

The present invention provides a method for removing a viral contaminantfrom a preparation, being a cell culture media or at least a componentof a cell culture media. The method comprises subjecting saidpreparation to filtration for at least about 24 hours through a virusfilter having an effective pore size of maximum 75 nm.

Further, the invention relates to the use of a virus filter having aneffective pore size of maximum 75 nm in a filtration for at least about24 hours for the removal of viral contaminant from a preparation, beinga cell culture media or at least a component of a cell culture media.

In addition, the invention relates to the use of a preparation, being acell culture media or at least a component of a cell culture mediaobtainable according to any method of the present invention for cellculture; pharmaceutical, diagnostic and/or cosmetic preparations as wellas in food preparations.

All methods and uses according to the invention can operate at avolumetric capacity of at least about 2000 L/m², preferably at leastabout 3000 L/m², most preferably at least about 5000 L/m². In addition,the preparation is subjected to filtration and the filtration isperformed, respectively, for at least 24 hours or for at least about 48hours up to about 7 months or about 72 hours up to about 3 months. Thefiltration is performed at a temperature from about 2° C. to about 60°C., or about 10 to about 40° C., preferably about 15 to about 37° C.

In all embodiments of the invention filtration is performed at apressure ranging from about 100 mbar to about 4000 mbar, preferably fromabout 200 mbar to about 3500 mbar, most preferably from about 1000 mbarto about 3000 mbar.

In all embodiments of the invention the used virus filter achieves atleast a 1 Log₁₀ reduction value (LRV) for a viral contaminant.

Surprisingly, it has been found that the volumetric capacity of virusfilters can be enormously increased when operating the filtrationprocess for at least about 24 hours. Normally, preparations for cellculture e.g. bulk cell culture media or buffers are filtered batchwisewithin about 2 to about 10 hours after preparation of the bulkpreparations in order to avoid contamination of the preparations bybacterial or viral growth. It has turned out that in practice themaximum capacity of the used virus filters is not nearly exploited infiltration processes filtering the respective preparations within atimeframe of about 2 to about 10 hours. Therefore excessive filter areahas to be used. In contrast thereto, it has been found that due to theuse of the method of the present invention, the volumetric capacity ofthe used costly virus filters can be better exploited leading to a 2 to100-fold increase of the volumetric capacity while maintaining thefilter integrity. Although a 2 to 3-fold increase of volumetric capacityalready has a great impact to the production process and the relatedproduction costs, with the method according to the invention an up to100-fold increase or even more can be achieved. This offers greatopportunities and makes viral removal cost efficient even with costlyvirus filters that now can be used to further improve viral safety incell culture processes, in particular in upstream viral removal of cellculture processes, pharmaceutical, diagnostic and/or cosmetic and foodprocesses.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainembodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

More particular descriptions of the invention are made by reference tocertain exemplary embodiments thereof which are illustrated in theappended Figures. These Figures form a part of the specification. It isto be noted, however, that the appended Figures illustrate exemplaryembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1A-1C is showing the virus filtration kinetics performed applying aflow controlled virus filtration (FIG. 1C) using different filters allcombined with cell culture media supplemented with 3 different soyhydrolysate lots (Kerry HyPep1510 #1, DOMO SE50 MAF UF #1 and #2).

Filter and experimental conditions applied (see also Example 1 toExample 4):Filter A: Sartorius Virosart CPV, 180 cm²; at 30° C. with flow rates ofabout 30 L/(m²×hr)Filter B: Millipore Viresolve NFP 3.1 cm²; at 30° C. with flow rates ofabout 40-60 L/(m²×hr). Filtrations were carried out for up to a maximumof 9 days or until a maximum pressure of 2000 mbar was exceeded. FIG. 1Ais showing the volumetric capacity as filtered volume per membranesurface area plotted against the time, ranging from about minimum 4000L/m² to about 12000 L/m². Maximum pressure at the end of filtration wasbetween about 600 mbar and 2400 mbar dependent on the filter type (FIG.1B). In general the difference between the soy hydrolysates isconsiderably low for the volumetric capacity and the maximum pressure.

FIG. 2A-2C is showing the virus filtration kinetics performed applying aflow controlled virus filtration (FIG. 2C) using different filters andcell culture media supplemented with 3 different soy hydrolysate lots(Kerry HyPep 1510 #1, DOMO SE50 MAF UF #1 and #2).

Filter and experimental conditions applied (see also Example 1 toExample 4):Filter A: Sartorius Virosart CPV, 180 cm²; at 30° C. with flow rates ofabout 30 L/(m²×hr)Filter D: Asahi BioEX 10 cm²; at ambient temperature (about 22° C.) withflow rates of about 20 L/(m²×hr). In contrast to the experimentsdescribed in FIG. 1, the filtrations were carried out for a longer timespan up to 81 days or until a pressure of 2000 mbar was reached. FIG. 2Ais showing the volumetric capacity as filtered volume per membranesurface area plotted against the time, ranging from about minimum 16000L/m² (for filter A with DOMO SE50 MAF #2) to about 35000 L/m² (forfilter D with all 3 different hydrolysate lots). Maximum pressure at theend of filtration was between about 1200 mbar and 2000 mbar dependent onthe filter type (FIG. 2B). In general the difference between the soyhydrolysates is considerably low for the volumetric capacity and themaximum pressure.

FIG. 3 is a graph showing the relationship between flux and differentialpressure as observed at about 22° C. using Filter A (Sartorius VirosartCPV 180 cm²) and media containing soy hydrolysate DOMO SE50 MAF UF, Lot#2 (see Example 5).

A minimum differential pressure of about 100 mbar is required to achievea minimum detectable specific flow rate, which is then graduallyincreasing with an obviously linear proportional correlation betweenspecific flow rate and differential pressure.

FIG. 4A-4C is showing the difference between a pressure controlled and aflow rate controlled filtration (FIG. 4A) using Filter A (Sartorius CPV,180 cm²) and medium with soy hydrolysate Kerry HyPep 1510 #2 (seeExamples 1 to Example 4). The filtrations were carried out for 19 daysand reached in this time a volumetric capacity of about 6000-7000 L/m².The final pressure of the flow controlled filtration was comparable tothe pressure of the pressure controlled filtration (see FIG. 4B), andthe final specific flow rate of the pressure controlled filtration wascomparable to the flow rate of the flow rate controlled filtration (seeFIG. 4C). This demonstrates that both control strategies for virusfiltration can result in comparable volumetric capacity.

FIG. 5A-5C is showing the results of a 10 L bioreactor experiment usingvirus filtered medium versus non virus filtered medium described inExample 6. Cell culture media were virus filtered batch wise with FilterA prior to start of the experiment. Experiments were carried out inparallel each using cell culture media supplemented with 3 different soyhydrolysates (Kerry HyPep 1510, Lot#1, DOMO SE50 MAF UF, Lot#1 and DOMOSE50 MAF UF, Lot#2). Data were calculated from the last 3 weeks of a 4week continuous cell culture. No differences between the respectivevirus filtered media (Soy 1 NF, Soy 3 NF and Soy 2 NF) versus theirunfiltered reference (Soy 1, Soy 3 and Soy 2) could be detected for thespecific productivity (FIG. 5A), the volumetric productivity (FIG. 5B)and the specific growth rate (FIG. 5C).

FIG. 6A-6C is showing the results of a 120 L bioreactor experiment usingvirus filtered medium versus non virus filtered medium described inExample 7. Cell culture media were virus filtered inline of the mediumfeed line of the bioreactors using alternatively Filter E (SartoriusVirosart CPV, 2000 cm²) and Filter F (Millipore Viresolve NFP 850 cm²)for about 58 days in continuous mode. Time intervals and volumetriccapacity of the virus filtered medium feed are shown in FIG. 6A. Datawere calculated for the intervals using the different filters. Nodifferences between the respective virus filtered media versus theunfiltered reference could be detected for the specific growth rate(FIG. 6B) and the volumetric productivity (FIG. 6C).

FIG. 7 shows the change of MMV infectivity titer [TCID₅₀/mL] found insequential filtrate samples taken in the course of the filtration of MMVspiked medium containing soy hydrolysate DOMO SE50 MAF#5 UF with FilterG (ASAHI Planova 15N) virus filters (see Example 8). Low level virusbreak-through was observed within 2 to 3 days. Nonetheless virus removalwas seen to be effective.

FIG. 8 shows the change of MMV infectivity titer [TCID₅₀/mL] found insequential filtrate samples taken in the course of the filtration of MMVspiked medium containing soy hydrolysate (Run #1 with soy hydrolysateDMV SE50 MAF UF #5; Run #2 with soy hydrolysate DMV SE50 MAF UF #4) withFilter D (Asahi BioEX) virus filters (see Example 9). No virusbreak-through was observed and virus removal was seen to be effectiveand complete.

FIG. 9 shows the change of MMV infectivity titer [TCID50/mL] found insequential filtrate samples taken in the course of the filtration ofMMV-spiked media as described in Example 10. No virus break-through wasobserved for the runs #1 and #2 (Filter D) resulting in effective andcomplete virus removal from the soy hydrolysate containing media.Low-level virus break-through was observed for the runs Runs #3 and #4(Filter I) and runs #5 and #6 (Filter G) resulting in effective but notcomplete virus removal from the soy hydrolysate containing media. Moresignificant virus break-through was observed for the runs #7 (Filter B)and run #8 (Filter H) resulting in reduced virus removal factors at thelimit of significance. However with all filters at least in oneexperiment a minimum overall titer reduction of more than about 1 logTCID50/mL could be achieved.

TABLE 1 Combination of virus filters and soy hydrolysates used inspiking experiments Overall reduction Soy hydrolysate factor Experiment# Filter Lot [log₁₀ (TCID₅₀/mL)] 1 D 7 >5.1 2 D 6 >4.6 3 I 3 4.5 4 I5 >5.4 5 G 7 4.4 6 G 7 4.1 7 B 6 1.5 8 H 3 1.2

FIG. 10 shows the kinetics of a viral filtration performed as describedin Example 11. Pressure was maintained between 0.8 and 1.2 bar (1.1 baraverage) except for the intentional pressure and flow interruptions,which simulate the worst case of operational conditions. Initial flowrates of about 38 L/(m²×hr) were achieved, which gradually decreaseduntil the end of the experiment, however, a minimum flow rate of 4L/(m²×hr) could be maintained. Overall duration including the pressureinterruptions was 30 days. Approximately 6500 L/m² were passed over thefilter.

FIG. 11 shows the change of MMV infectivity titer [log₁₀(TCID₅₀/mL)]found in sequential filtrate samples taking in the course of thefiltration of MMV-spiked media as described in example 11. No virusbreak-through was observed in any of the 20 fractions assayed. Virusloads ranged from <0.9 [log₁₀(TCID₅₀)] to <2.8 [log₁₀(TCID₅₀)] dependingon fraction volume. The total virus load in the filtrates was <3.0[log₁₀(TCID₅₀)] which—when subtracted from the initial virus load of thespiked material (i.e. 8.5 [log₁₀(TCID₅₀)])—results in an overall virusreduction factor of >5.5 log₁₀. This was seen to be effective andcomplete.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for removing a viral contaminantfrom a preparation, being a cell culture medium or at least a componentof a cell culture medium. The method comprises subjecting saidpreparation to filtration for at least about 24 hours through a virusfilter having an effective pore size of maximum 75 nm.

Further, the invention relates to the use of a virus filter having aneffective pore size of maximum 75 nm in a filtration for at least 24hours for the removal of viral contaminant from a preparation, being acell culture medium or at least a component of a cell culture medium.

In addition, the invention relates to the use of a preparation, being acell culture medium or at least a component of a cell culture mediumobtainable according to any method of the present invention for cellculture; pharmaceutical, diagnostic and/or cosmetic preparations as wellas in food preparations.

In all embodiments of the invention the preparation is subjected tovirus filtration, the virus filtration or the use of the virus filter isperformed for at least about 24 hours, about 48 hours, about 72 hours,about 4 days, about 5 days, about 6 days, about 7 days, about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2months, about 3 months, about 4 months, about 5 months, about 6 monthsor about 7 months. Further in one embodiment the preparation issubjected to virus filtration or the virus filtration is performed forabout 1 week to about 3 weeks, about 2 weeks to about 3 weeks, about 1week to about 4 weeks about 2 weeks to about 4 weeks, about 1 week toabout 7 months, about 1 months to about 5 months, about 2 months toabout 5 months, about 2 months to about 4 months, about 2 months toabout 3 months or at least about 24 hours up to about 7 months or about48 hours up to about 5 months or about 72 hours up to about 3 months.Further, in one embodiment the preparation is subjected to virusfiltration or the virus filtration is performed for longer than about 48hours up to about 7 months, preferably about one week to about 5 monthsor about 3 weeks to about 3 months or about 2 months to about 3 months.

The method according to the invention can operate at a volumetriccapacity of at least about 2000 L/m², or at least about 3000 L/m², or atleast about 4000 L/m², or at least about 5000 L/m², at least about 7500L/m², at least about 10000 L/m², or at least about 20000 L/m². In thisrespect the “volumetric capacity” refers to the volume of solution thatcan be filtered through a specified area of the virus filter membranebefore filtrate flow is reduced or the back pressure is increased toundesirable operating conditions due to the clogging of the filtermembrane.

It is contemplated that the present invention including all embodimentscan be employed alone or in conjunction with other approaches known inthe art for minimizing viral contamination, e.g. screening, sourcing,detection, viral inactivation, adsorptive retention, etc. The presentmethods target the entry of unwanted viral agents through thepreparation, being a cell culture medium or at least a component of acell culture medium, early in the production process and provide a viralreducing mechanism. Advantages of the present invention include ease ofimplementation on a large-scale basis, reduced filter membrane areaneeded to process a given volume of a preparation, being a cell culturemedium or at least a component of a cell culture medium, the reducedcost ensuing therefrom. In particular virus filtration of preparationsaccording to the invention is easy to integrate into continuousmanufacturing processes, e.g. continuous cell culture processes likeperfusion or chemostat like bioreactor systems.

The term “temperature” as used herein concerns the temperature of thefiltered preparations according to the invention, e.g. a cell culturemedium or buffer, at the time it passes through the virus filter. In oneembodiment according to the invention the temperature ranges from about2° C. to about 60° C. In one embodiment the lower limit of thetemperature range is about 2° C., about 4° C., about 8° C., about 10°C., about 15° C., about 20° C., about 22° C., about 25° C., about 30°C., about 37° C. or about 40° C. The upper limit of the temperaturerange according to the invention is about 10° C., about 20° C., about22° C., about 25° C., about 30° C., about 37° C., about 40° C., about50° C. or about 60° C. In one embodiment the temperature is in a rangefrom about 4° C. to about 45° C., or at a temperature range from about10° C. to about 40° C., or from about 20° C. to about 40° C., or fromabout 30° C. to about 37° C. Also in one embodiment the temperature isambient temperature that is a range from about 20° C. to about 30° C. Ofcourse, also embodiments are preferred where the preparations aresubjected to filtration without any further heating or cooling of thepreparations. Therefore, in a further embodiment a temperature of about10° C. to about 30° C. is used depending from the temperature of therespective place at which the filtration is performed. In anotherembodiment, temperatures of about 30° C. to about 37° C. are used, e.g.by preheating of the liquid preparation prior to filtration. Thefiltrate resulting from this filtration of a preparation can becontinuously fed to a bioreactor.

In one embodiment of the invention filtration is performed at a pressureranging from about 100 mbar to about 4000 mbar, or from about 200 mbarto about 3500 mbar. In one embodiment, virus filtration is performed ata pressure range, wherein the lower limit is about 100 mbar, about 200mbar, about 500 mbar, about 1000 mbar, about 1200 mbar, about 1500 mbar,about 2000 mbar, about 2500 mbar or about 2800 mbar. The upper limit isabout 1200 mbar, about 1500 mbar, about 2000 mbar, about 2500 mbar,about 2800 mbar or about 3000 mbar. In one embodiment, filtration isperformed at a pressure ranging from about 1000 to about 4000 mbar,about 1500 to about 3500 mbar, 1700 mbar to about 3300 mbar or about1000 mbar to about 2000 mbar.

Temperature and pressure adjustments may be utilized in furtherembodiments of the invention to regulate the specific flow rate and thevolumetric capacity. Further improvements in the volumetric capacity andthe time span of use of the virus filter can be obtained by regulatingother process parameters, such as filtration pressure and temperature.For instance, it has turned out that in some embodiments it is preferredto subject the preparation to filtration at a temperature of about 10°C. to about 40° C. at a pressure of about 1000 mbar to about 2000 mbar.

Preliminary filtration experiments have demonstrated the influence oftemperature of the preparations to be filtered on the specific flowrate. An about 50 to about 100% increase of flow rate was observed whenincreasing the filtration temperature from a preparation according tothe invention having a storage temperature of about 4° C. totemperatures of from about 18° C. to about 37° C. However, all theseembodiments are within the scope that the use of filtration for at leastabout 24 hours effects that the capacity of the used costly virusfilters can be better exploited leading to an 2 to 100-fold increase ofthe volumetric capacity while maintaining the filter integrity.

In one preferred embodiment the method for removing a viral contaminantfrom a preparation, being a cell culture medium or at least a componentof a cell culture medium, comprises the step of subjecting saidpreparation to filtration for at least about 10 days to about 2 monthsthrough a virus filter having an effective pore size of maximum 75 nm ata pressure of about 1000 mbar to 2000 mbar and a temperature of 10° C.to 40° C. having a volumetric capacity of at least 2000 L/m². Of course,all other parameters can be combined also with this embodiment. Inaddition, as a further preferred embodiment said method is performed ina continuous filtration mode, wherein the preparation is preferred acell culture medium, e.g a cell culture medium comprising a soyhydolysate or a cell culture medium comprising animal derivedcomponents, wherein the filtrate is continuously fed to a bioreactor, inparticular a chemostat reactor. In another embodiment this embodimentcan further be performed using at least 2 virus filters arranged inparallel or in series.

It is contemplated that the virus filtration methods as described hereincan be used to reduce viral contamination from any preparation being acell culture medium or a component of a cell culture medium, i.e. amedium and buffers suitable for growth of animal cells, and preferablymammalian cells, in in vitro cell culture. Typically, culture mediumcontains a buffer, salts, energy source, amino acids, vitamins and traceessential elements.

The term “preparation” also includes any component being a possible partof a cell culture medium in the sense of the present invention andcapable of supporting growth of the appropriate cell in culture. Saidpreparations include e.g. a buffer or solutions of at least one aminoacid or protein; solutions of at least one vitamin; solutions of atleast one organic or inorganic salt; or solutions comprising at leastone source of carbohydrates or sugars.

In the context of the present invention “Log reduction value” (LRV) is ameasure of a membrane's efficiency in retaining a particle such asbacteria or virus, defined as the logarithm (base 10) of the ratio ofsaid particle count in the feed stream to the particle count in thevirus filter membrane permeate. The LRV value is specific to a giventype of particle. In one embodiment according to the invention the virusfilter achieves at least a 1 Log₁₀ reduction value (LRV) for a viralcontaminant, or at least a 2 Log₁₀ reduction value (LRV) for a viralcontaminant, or at least a 3 Log₁₀ reduction value (LRV) for a viralcontaminant, or at least a 4 Log₁₀ reduction value (LRV) for a viralcontaminant, or at least a 5 Log₁₀ reduction value (LRV) for a viralcontaminant, or at least a 6 Log₁₀ reduction value for viralcontaminant, or at least a 7 Log₁₀ reduction value for viralcontaminant, or at least a 8 Log₁₀ reduction value for viralcontaminant, preferably at least a 4 Log₁₀ reduction value (LRV) for aviral contaminant. Of course, it is evident for a skilled person in theart, that any Log₁₀ reduction value (LRV) of a viral or potential viralcontaminant of the preparation to be filtered is beneficial in order toimprove the safety of a production process. Therefore, especially thisparameter can be combined with all other parameters that are used in themethod of the present invention.

“Flux,” as used herein is interchangeable used with “specific flow rate”or “flow rate” is a measure used to characterize membranes, refers tothe rate of filtrate flow (expressed in the volume or weight of solutionthat permeates through the virus filtration membrane per filter area andtime, e.g. L/(m²×hr). In the context of the invention the term“specific” means within a defined time, however, when only “flow rate”is used, also from the units of this parameter it is evident that the“specific flow rate” is meant. As abbreviation of the quantity “volume”given in the unit “litre” “I” oder “L” is used interchangeably. Thespecific flow rate within the method of the present invention may varywithin a range or remain substantially fixed throughout the duration ofthe filtration process using a given virus filter. In one embodiment ofthe present invention the specific flow rate may range from about 5L/(m²×hr) to about 500 L/(m²×hr) for at least 24 hours up to about 7months. The lower limit for the flux may be about 5 L/(m²×hr) or about10 L/(m²×hr). The upper limit may be about 25 L/(m²×hr), about 75L/(m²×hr), about 100 L/(m²×hr), about 200 L/(m²×hr), about 250L/(m²×hr), about 300 L/(m²×hr) or about 500 L/(m²×hr). The flux mayfurther range from about 5 L/(m²×hr) to about 100 L/(m²×hr), about 10L/(m²×hr) to about 100 L/(m²×hr) or about 10 L/(m²×hr) to about 25L/(m²×hr).

“Batch filtration,” otherwise known as “batch wise filtration” orfiltration done in batch mode, refers herein to a process wherein aspecific total amount or volume of a preparation, being a cell culturemedium or at least a component of a cell culture medium, is filteredthrough a virus filter in one batch dependent on the capacity of thevirus filter and wherein the filtration process is finalized before thefiltrate is directed or fed to the process in which it is used orconsumed.

The term “continuous filtration” or “online filtration” or “in linefiltration” refers to a filtration process, wherein the specific totalamount or volume of a preparation, being a cell culture medium or atleast a component of a cell culture medium, is filtered through thevirus filter continuously dependent on the capacity of the virus filterand wherein the filtration process is still going on when the filtrateis already directed or fed to the process in which it is used orconsumed.

All embodiments of the present invention may be performed using batch orcontinuous filtration. The beneficial effect of the invention is alreadyachieved by subjecting a preparation, being a cell culture medium or atleast a component of a cell culture medium, to filtration for at least24 hours through a virus filter having an effective pore size of maximum75 nm in order to remove a viral contaminant from said preparation.

In a preferred embodiment according to the invention the method forremoving a viral contaminant from a preparation, being a cell culturemedium or at least a component of a cell culture medium, wherein saidpreparation is subjected to filtration for at least about 24 hoursthrough a virus filter having an effective pore size of maximum 75 nm,is performed as continuous filtration. This mode of operation has theadvantage that the produced filtrate of the preparation can be directlyand continuously fed to the process where it is used or consumed. In afurther preferred embodiment the virus filtered preparation, being acell culture medium or at least a component of a cell culture medium,can directly and continuously feed a bioreactor, more preferably a largescale bioreactor used in a continuously fed cell culture process, e.g. achemostat process, a perfusion process or a fed batch process. Thisembodiment is performed in one embodiment using a pressure of about 1000mbar to 2000 mbar and a temperature of 10° C. to 40° C., wherein thevolumetric capacity is at least 2000 L/m² or at least 5000 L/m². Inaddition, it is further preferred that the virus filtration of thepreparation is performed for at least about 24 hours or about 48 hoursup to about 7 months, more preferred for at least about one week up toabout 5 months and most preferred for at least about one to about 3weeks or about 3 weeks to about 3 months and even most preferred atleast about 2 to about 3 months. Of course, all other parameters can becombined with this embodiment. In addition, it is preferred that thepreparation to be filtered is a cell culture medium and the mode offiltration is a continuous mode filtration.

Of course, it is known by a skilled person in the art that the virusfiltered preparations obtainable according to any of the methodsaccording to the invention may also be directed or fed to otherproduction processes relating to cell culture; pharmaceutical,diagnostic and/or cosmetic preparations as well as food preparations.Also in those embodiments a continuously filtration of the preparationsis preferred.

Hence, the invention also relates to the use of a preparation, being acell culture medium or at least a component of a cell culture mediumobtainable according to any of the methods according to the inventionfor cell culture; pharmaceutical, diagnostic and/or cosmeticpreparations as well as in food preparations.

In some embodiments, the cell culture medium to be virus filtered issterile, or otherwise pretreated. In some embodiments the cell culturemedium comprises animal proteins or serum or other animal derivedcomponents, or is animal protein-free, or serum-free, or free of animalderived components, or possess any combination of the foregoingcharacteristics. In other embodiments, the cell culture medium comprisesvarying concentrations and species of plant or microbial derivedhydrolysates, especially soy hydrolysates. In a preferred embodiment thecell culture medium is an animal protein-free medium comprising varyingconcentrations of at least one soy hydolysate. However, it has to beemphasized that the method according to the invention is especiallysuitable for virus filtration of preparations comprising animal proteinor serum or other animal derived components in order to further improvethe virological safety of those preparations, in particular when used incell culture processes, in pharmaceutical, diagnostic and/or cosmeticpreparations as well as in food preparations.

“Cell culture medium” is defined, for purposes of the invention, as amedium suitable for growth of cells, and preferably animal cells, morepreferably mammalian cells, in in vitro cell culture. Any medium capableof supporting growth of the appropriate cells in cell culture can beused. The cell culture medium according to the invention may be based onany basal medium such as DMEM, Ham's F12, Medium 199, McCoy or RPMIgenerally known to the skilled worker. The basal medium may comprise anumber of ingredients, including amino acids, vitamins, organic andinorganic salts, and sources of carbohydrate, each ingredient beingpresent in an amount which supports the cultivation of a cell which isgenerally known to the person skilled in the art. The medium may containauxiliary substances, such as buffer substances like sodium bicarbonate,antioxidants, stabilizers to counteract mechanical stress, or proteaseinhibitors. If required, a non-ionic surfactant such as mixtures ofpolyethylene glycols and polypropylene glycols (e.g. Pluronic F68®,SERVA) can be added as a defoaming agent.

As used herein, an “animal protein-comprising medium” is a cell culturemedium that comprises any protein that has been derived from a humansource or an animal source.

As used herein, a “protein-free medium” is a cell culture medium that isfree of any protein that has been derived from a human source or ananimal source.

The term “animal protein-free cell culture medium” according to theinvention refers to a medium that does not contain proteins and/orprotein components from higher multicellular non-plant eukaryotes.Typical proteins that are avoided are those found in serum andserum-derived substances, such as albumin, transferrin, insulin andother growth factors. The animal protein free cell culture medium isalso free of any purified animal derived products and recombinant animalderived products as well as protein digests and extracts thereof orlipid extracts or purified components thereof. Animal proteins andprotein components are to be distinguished from non-animal proteins,small peptides and oligopeptides obtainable from plants (usually 10-30amino acids in length), such as soy bean, and lower eukaryotes, such asyeast which may be included into the animal protein free cell culturemedium according to the invention.

The term “hydrolysate” includes any digest of an animal derived or plantderived source material or extracts derived from yeast or bacteria. Inthe cell culture medium according to the invention “soy hydrolysate” canbe comprised that may be a highly purified soy hydrolysate, a purifiedsoy hydrolysate or crude soy hydrolysate.

The term “serum-comprising” as applied to medium includes any cellculture medium that does contain serum.

The term “serum-free” as applied to medium includes any cell culturemedium that does not contain serum. By “serum free”, it is understoodthat the medium has preferably less than 0.1% serum and more preferablyless than 0.01% serum. The term “serum” refers to the fluid portion ofthe blood obtained after removal of the fibrin clot and blood cells.

In some embodiments, the filtrate or the flow of the filtrate obtainedfrom the filtration process is fed to a large-scale cell culture andbioreactor respectively. A “large-scale” cell culture, as used herein,refers to a cell culture at a scale of at least about 100 L, at leastabout 200 L, at least about 300 L, at least about 400 L, at least about500 L, at least about 1000 L, at least about 1500 L, at least about 2000L, at least about 2500 L, at least about 3000 L, at least 4000 L, atleast about 5000 L, at least about 7500 L, at least about 10000 L or atleast about 20000 L. In a preferred embodiment the filtrate flowobtained in any method according to the invention is fed to a bioreactorused in a chemostat process, a perfusion process or a fed batch process,preferably by continuous filtration.

The cell culture contemplated herein may be any cell cultureindependently of the kind and nature of the cultured cells and thegrowth phase of the cultured cells, e.g. adherent or non-adherent cells;growing, or growth-arrested cells.

The term “sterile,” as used according to the invention, refers to asubstance that is free, or essentially free, of microbial and/or viralcontamination. In this respect the “contaminant” means a material thatis different from the desired components in a preparation being a cellculture medium or at least a component of a cell culture medium. In thecontext of “sterile filtration”, the term sterile filtration is afunctional description that a preparation is filtered through a sterilefilter to remove bacterial and/or mycoplasma contaminants.

The term “virus filtration” is used herein interchangeably with the term“nanofiltration” and means that for the filtration process a virusfilter is used having a defined effective pore size. In general thosefilters are dedicated to remove viruses.

The viral contaminant targeted for removal by filtration according toall methods of the invention may be any virus presently known in the artor to be discovered in the future. This definition also includes apotential viral contaminant to be removed by filtration and alsoincludes that more than one virus is removed by the methods of thepresent invention. For example, the viral contaminant or potential viralcontaminant can be a member of the viral families of Orthomyxoviridae,Arenaviridae, Paramyxoviridae, Rhabdoviridae, Coronaviridae,Flaviviridae, Picornaviridae, Togaviridae, Arteriviridae,RetParvoviridae, Bunyaviridae, Caliciviridae, Retroviridae, Reoviridae,Circoviridae, Adenoviridae, Poxviridae, Herpesviridae, Iridoviridae orReoviridae. More specifically, the viral contaminant may be any of thegroup consisting of canine parvoviridae (CPV), minute virus of mice(MVM), Cache Valley virus, Bunyamwera virus, Northway encephalitisvirus, Influenza A/B virus, Junin virus, Parainfluenza virus 1/2/3,Simian virus 5, Mumps virus, Bovine respiratory syncytial virus, Sendaivirus, Newcastle disease virus, Pneumonia virus of mice, vesicularstomatitis virus, Rabies virus, Bovine coronavirus, Murine hepatitisvirus, Yellow fever virus, West Nile virus, Dengue virus, Tick borneencephalitis virus, St. Louis encephalitis virus, Vesivirus 2117,Encephalomyocarditis virus, Coxsackie virus B-3, Theiler's mouseencephalitis virus, Foot and mouth disease virus, Bovine enterovirus,Porcine enterovirus, Semliki Forest virus, Sindbis virus, Rubella virus,Japanese encephalitis virus, Eastern equine encephalitis virus, Porcinereproductive and respiratory syndrome virus, Foamy virus, Reovirus1/2/3, Avian reovirus, Rotavirus, Porcine circovirus 1, Adenovirus,Pseudorabies virus, Murine gammaherpes 68, Herpes simplex virus 1, Frogvirus 3, minute virus of mice-cutter (MVMc), bluetongue virus (BTV),Epizootic haemorrhagic disease virus (EHDV), bovine viral diarrhea virus(BVDV), porcine parvovirus (PPV), encephalomyocarditis virus (EMCV),Reovirus 3, and murine leukemia virus (MuLV), Hepatitis A, polio, orParvoviridae B19.

The term “virus filter,” is used interchangeably herein with the terms“virus-retentate filter”, “viral filter” and “nanofilter” and refersgenerally to a filter which characteristics as a whole make it suitablefor virus retention having an effective pore size to fulfill thisfunction. These characteristics include, by way of example, membraneattributes such as morphology, pore shape, pore density, and uniformity,effective pore size, membrane thickness, etc. The virus filter membranesuseful in the present invention encompass membranes that operate by sizeexclusion and charge, possibly in combination with adsorptive retention.Size exclusion and adsorptive retention mechanisms are not necessarilyexclusive of one another and a filter may well employ one or moremechanisms.

The virus filter as defined in the present invention and used in oneembodiment of the present invention is characterized by having amembrane with an effective pore size of maximum 75 nm. In one embodimentaccording to the invention the lower limit of the effective pore size isabout 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30nm, about 35 nm, about 50 nm or about 60 nm. In said embodimentaccording to the invention the upper limit of the effective pore size isabout 10 nm, about 15 nm, about 20 nm, about 25 nm, about 35 nm, about50 nm, about 60 nm or about 75 nm. In some embodiments of the invention,the virus filter has an effective pore size from about 5 to about 75 nmor from about 10 to about 75 nm or from about 15 to about 75 nm or fromabout 20 to about 75 nm or from about 15 to about 50 nm or from about 15to about 35 nm.

Effective pore size, as used herein, is a characteristic of a membraneand refers to the size of a particle which can be effectively retainedby the membrane, considering that the level of effectiveness isdescribed by a logarithmic reduction factor of a particle of such size.

The virus filter used in the methods of the present invention can be anyfilter having a construction sufficient to withstand a volumetriccapacity of at least about 2000 L/m², or at least about 3000 L/m², or atleast about 4000 L/m² or at least about 5000 L/m², or at least about7500 L/m², or at least about 10000 L/m² or at least about 20000 L/m², orwhich can be operated for a time span of more than about 24 hours up toabout 7 months or preferably for at least about 48 hours, about 72hours, about 4 days, about 5 days, about 6 days, about 7 days, about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about2 months, about 3 months, about 4 months, about 5 months, about 6 monthsor about 7 months.

Of course, if more than one filter is used in a method according to theinvention also different types of virus filters can be used and combinedin the filtration process, preferably in parallel or in series.

Exemplary virus filter comprise a single or multilayer membrane and areconstructed of material such as polyvinylidene fluoride (PVDF),cellulose, modified cellulose, e.g. cuprammonium regenerated cellulosehollow fibers or polyethersulfone. The membranes of the virus filtersmay have a neutral, negative, or positive charge. The membranes may beionic membranes, i.e. they may contain cationic or anionic groups, butneutral membranes may be preferred depending on the pH conditions. Thevirus filter membranes may be selected from hydrophobic and hydrophilicmembranes. In a preferred embodiment the membrane of the virus filterused in the method according to the invention is made frompolyvinylidene fluoride (PVDF) or polyethersulfone.

Manufacturers of exemplary filters having demonstrated ability to removeviruses include, without exclusion, Asahi/Planova, PALL, Millipore,Sartorius, Gambro, and Amersham/AG Technology. Filters suitable for usein the present invention include, without limitation, Asahi's Planova 15N filter (Asahi Kasei Corporation, Planova Division), Planova 20 Nfilter (Asahi Kasei Corporation, Planova Division), Planova 35 N filter(Asahi Kasei Corporation, Planova Division), and the BioEX filter (AsahiKasei Corporation, Planova Division).

Of course, it is desirable that the filter used in one of the methods ofthe present invention is autoclavable and/or autoclaved and/or otherwisesterilized before use. However, all other possibilities to ensure thesterility of the used virus filter are suitable to perform theinvention. Furthermore it is desirable that the filter can be integritytested prior to use and/or after use. In a preferred embodiment, in themethod according to the invention an autoclaved, integrity tested virusfilter is used having a membrane of polyvinylidene fluoride (PVDF) orpolyethersulfone.

“Filtrate”, used interchangeably herein with the term “permeate,” refersto the solution that crosses a filter or membrane as well as thesolution that has crossed a filter or membrane.

“Retentate”, as used herein, refers to the component of the solutionthat is retained and does not cross a filter or membrane as well as thathas not crossed a filter or membrane.

The virus filtration equipment useful in the present invention comprisesat least one virus filtration membrane element dividing the feed into apre and post filter section. The filtration equipment typically alsoincludes means for controlling the pressure and flow, such as pumps andvalves and flow and pressure meters and density meters. The equipmentmay also include several filtration membrane elements in differentcombinations, arranged in parallel or series or both.

The filtration flux varies in accordance with the pressure. In general,at a normal operation range, the higher the pressure, the higher theflux. The flux also varies with the temperature. An increase of theoperating temperature increases the flux. However, with highertemperatures and with higher pressures there is an increased tendencyfor a membrane rupture. For inorganic membranes, higher temperatures andpressures and higher pH ranges can be used than for polymeric membranes.

For a skilled person in the art it is unambiguously evident that insteadof a virus filter according to the invention a filter can be used havinga molecular weight cut-off of less than about 5000 Daltons or less thanabout 1000 Daltons in order to remove also viruses. In this context“Molecular weight cut-off” (MWCO) is a membrane characteristic of afilter that specifies the average molecular weight of solutes, howeveralso particles or viruses will not permeate the membrane of this filter.

The pH value in the virus filtration process of the present inventioncan be set at any range necessary to preserve the stability andfunctionality of the preparation being filtered, preferably a cellculture medium or buffer. For example, the pH value may be set at about1 to about 10, preferably about 2 to about 8 or about 3 to about 7,preferably about 6.8 to about 8 or most preferably at about 7.0 to about7.45 (physiological pH value).

It is also contemplated that the method according to the invention maybe integrated into a system downstream of a sterilizing grade filterthat removes bacteria contaminant and thereby yield a sterile feedstream of a preparation that can be the “starting preparation”, i.e. thepreparation being used in any method according to the invention.

In one embodiment the method of the invention may be performed using twoor more filters arranged in series. This has the advantage of augmentingvirus clearance capacity and safeguard against potential virus filterfailure or breakthrough. In alternative embodiments, filtration isperformed using two or more virus filters arranged in parallel, therebypermitting virus filter replacement without disrupting a continuousprocess and preventing unforeseen medium holds, e.g. due to clogging.

In still other embodiments, filtration is performed using at least twofilters arranged in parallel in a piping system comprising a Y-shapedjunction, wherein each filter is in fluid communication with a branch ofthe Y-shaped junction and a preparation supply source. In someembodiments, the Y-shaped junction comprises a connector. In otherembodiments, filtration is performed using a setup containing aplurality of filters arranged both in series and in parallel. Especiallyuseful in the context of the present invention is an arrangement,wherein at least a second filter is arranged in parallel in connectionwith other parallel filters or filters arranged in series in order tohave the possibility to replace one of the filters without stopping thefiltration process for maintenance reasons.

In some embodiments, the filter is subjected to an integrity test priorto use. The integrity test may take the form of an air-water diffusionbased test, wherein air is directed to the filter and the filter thensubmerged in sterile water and examined for bubbles, which wouldindicate a leak in the filter.

In one embodiment, the virus filter or the virus filter membrane may bepretreated before the virus filtration procedure, e.g. by washing with awashing agent, in particular with an acidic washing agent, an alkalinewashing agents and/or ethanol.

In one embodiment of the invention also tangential flow filtration maybe performed in the method according to the invention. In the context ofthe present invention “tangential flow filtration,” is usedinterchangeably herein with the term “crossflow filtration.” Intangential flow mode, the liquid flow path on the upstream side of thefilter is directed roughly parallel to or tangential to or across thefilter surface. Passage of the permeate is facilitated by restrictingthe flow of retentate relative to feed, resulting in backpressure to thesystem and permitting permeate migration through the filter membrane.The constant sweeping current across the membrane surface has the effectof minimizing clogging by contaminants in the product being filtered.Any virus filter is suitable that achieves at least a 1 Log₁₀ reductionvalue (LRV) for a viral contaminant, or at least a 2 Log₁₀ reductionvalue (LRV) for a viral contaminant, or at least a 3 Log₁₀ reductionvalue (LRV) for a viral contaminant, or at least a 4 Log₁₀ reductionvalue (LRV) for a viral contaminant, or at least a 5 Log₁₀ reductionvalue (LRV) for a viral contaminant, or at least a 6 Log₁₀ reductionvalue for viral contaminant, or at least a 7 Log₁₀ reduction value forviral contaminant, or at least a 8 Log₁₀ reduction value for viralcontaminant, preferably at least a 4 Log₁₀ reduction value (LRV) for aviral contaminant. All log reduction factors may apply for any of theeffective pore sizes of maximum 75 nm of the virus filter. In oneembodiment according to the invention the lower limit of the effectivepore size of the virus filter is about 5 nm, about 10 nm, about 15 nm,about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 50 nm or about60 nm. In said embodiment according to the invention the upper limit ofthe effective pore size of the virus filter is about 10 nm, about 15 nm,about 20 nm, about 25 nm, about 35 nm, about 50 nm, about 60 nm or about75 nm. In some embodiments of the invention, the virus filter has aneffective pore size from about 5 to about 75 nm or from about 10 toabout 75 nm or from about 15 to about 75 nm or from about 20 to about 75nm or from about 15 to about 50 nm or from about 15 to about 35 nm.

In some embodiments of the present invention normal flow filtration isused. “Normal flow filtration”, used interchangeably herein with theterms “dead end,” “single pass,” and “direct flow filtration,” refers toa virus filter filtration process wherein the liquid flow path isdirected usually perpendicular to the filter surface, dependent on theconstruction of the filter module the fluid stream could also bedirected tangential to the filter membrane, however in contrast tocrossflow filtration, no recirculation of retentate is applied, whichmeans that the specific flow rate before and after the filter isidentical. Any virus filter is suitable that achieves at least a 1 Log₁₀reduction value (LRV) for a viral contaminant, or at least a 2 Log₁₀reduction value (LRV) for a viral contaminant, or at least a 3 Log₁₀reduction value (LRV) for a viral contaminant, or at least a 4 Log₁₀reduction value (LRV) for a viral contaminant, or at least a 5 Log₁₀reduction value (LRV) for a viral contaminant, or at least a 6 Log₁₀reduction value for viral contaminant, or at least a 7 Log₁₀ reductionvalue for viral contaminant, or at least a 8 Log₁₀ reduction value forviral contaminant, preferably at least a 4 Log₁₀ reduction value (LRV)for a viral contaminant. All log reduction factors may apply for any ofthe effective pore sizes of maximum 75 nm of the virus filter. In oneembodiment according to the invention the lower limit of the effectivepore size of the virus filter is about 5 nm, about 10 nm, about 15 nm,about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 50 nm or about60 nm. In said embodiment according to the invention the upper limit ofthe effective pore size of the virus filter is about 10 nm, about 15 nm,about 20 nm, about 25 nm, about 35 nm, about 50 nm, about 60 nm or about75 nm. In some embodiments of the invention, the virus filter has aneffective pore size from about 5 to about 75 nm or from about 10 toabout 75 nm or from about 15 to about 75 nm or from about 20 to about 75nm or from about 15 to about 50 nm or from about 15 to about 35 nm.

As those of ordinary skill in the art would appreciate, all embodimentsof the invention can be implemented with the aid of any available systemtechnically useful for the purpose, e.g. a variable-speed or fixed-speedperistaltic pump, a centrifugal pump, etc. Any kind of pressurizedvessel or other container can be used to generate flow through the virusfilter with constant or variable pressure during the filtration process.

Those of ordinary skill in the art will appreciate that the choice offilter type and mode (dead end filtration or tangential flow filtration)will depend on factors such as composition, the protein content, themolecular weight distribution, impurity/particulate load or any otherbiochemical or physical property in the feed to be processed, processrequirements and limitations (allowable pressure, process time, volumesto be filtered) or characteristics of the potential viral contaminant,e.g. virus size. Availability of an in-process integrity test andlogistics of viral clearance studies must also be taken intoconsideration. Dead end filtration should typically be employed for feedstreams of high purity to yield a reasonable process flux whereas insome embodiments tangential flow filtration can accommodate feed streamswith high particulate load. In some preferred embodiments normal flowfiltration is preferred in combination with a continuous filtration modeusing a least one virus filter having an effective pore size of maximum75 nm. Of course, also this embodiment can be combined with all otherparameters of the present invention.

Of course, it is to be understood that this invention is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided might be different from the actual publicationdates that may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

Example 1: Scale Down Virus Filtration with Different Virus Filters andCell Culture Media

Virus filtration membranes from different manufacturers (see Table 2)were assessed for their filtration kinetics in different filter sizeswith cell culture media containing 4 g/L concentration of soyhydrolyates from different lots and suppliers (see Table 3). Cellculture media composition and preparation is described in Example 2.Filtration experiments were carried out either by controlling thepressure with a pressurized vessel (FIG. 3, FIG. 4, FIG. 5 and spikingexperiments in FIG. 7, FIG. 8 and FIG. 9), or by controlling the flowrate e.g. by a peristaltic pump (FIG. 1, FIG. 2, FIG. 3 and FIG. 6).Other equipment used for temperature and pressure control of theexperiments is described in Table 4.

TABLE 2 List of virus filters Internal filter code in Figures/ExamplesManufacturer/Product name/Size Filter A Sartorius Virosart CPV 180 cm²Filter B Millipore Viresolve NFP 3.1 cm² Filter C Pall Ultipor VF gradeDV20 700 cm² Filter D Asahi BioEX 10 cm² Filter E Sartorius Virosart CPV2000 cm² Filter F Millipore Viresolve NFP 850 cm² Filter G Asahi 15N 10cm² Filter H Pall Ultipor VF grade DV20 9.6 cm² Filter I SartoriusVirosart CPV 5 cm²

TABLE 3 List of soy hydrolysates Internal soy hydrolysate codeManufacturer/Product name/Internal in Figures/Examples lot number Soyhydrolysate 1 Kerry HyPep 1510 #1 Soy hydrolysate 2 DOMO SE50 MAF UF #1Soy hydrolysate 3 DOMO SE50 MAF UF #2 Soy hydrolysate 4 DOMO SE50 MAF UF#3 Soy hydrolysate 5 Kerry HyPep 1510 #2 Soy hydrolysate 6 DOMO SE50 MAFUF #4 Soy hydrolysate 7 DOMO SE50 MAF UF #5

TABLE 4 List of equipment WM Marprene bore mm × wall mm 3.2 × 1.6 and1.6 × 1.6 (Watson Marlow) Peristaltic pumps Watson Marlow 101U/R (WatsonMarlow) Pressure Vessel Sartorius Model 17532 (Sartorius-Stedim)Pressure Transducers: Pascal Ci CL1010 (Labom) and KrosFlo ACPM- 499-03N(Spectrum Labs) Balance Sartorius FBG64EDE-SOCE (Sartorius Stedim) WaterBath Haake DC10 (Thermo Scientific) Temperature sensor CEM IR-68Flexible InfraRed Thermometer

Example 2: Cell Culture Media Preparation

A general description of the cell culture media composition is providedin Table 5 below, with the composition of the different soy hydrolysateslisted in Table 3 above. The different batches of cell culture mediawere sterile filtered with a sterile grade filter, e.g. a PallFluorodyne® II DJL Membrane Filter Cartridge 0.1μ prior to the differentvirus filtrations described in the examples. Media preparationsdescribed here were used for all experiments described and shown in FIG.1 to FIG. 11.

TABLE 5 Media composition Concentration Component [g/kg] DMEM/HAMS F1211.76 Ethanolamine 0.00153 Lutrol F68 0.25 Soy hydrolysate 4.0 Traceelement - stock solution Max. 4 μg/L L-Glutamine 0.6 NaHCO₃ 2.0 Purifiedwater Ad 1 kg

Example 3: Filter Preparation

The virus filters were prepared according to product manuals of virusfilter manufacturers. Unless filters were delivered and assembledsterile, filters were autoclaved at >121° C. for 20 minutes.

Example 4: Integrity Test

After usage, filters of appropriate size (Filters A, C, E and F of Table2) were washed according to the respective manufacturer'srecommendations. A Forward Flow Test was performed with PalltronicFlowstar XC (Pall, US) according to the manufacturer's specifications.All integrity tests performed after filtration experiments describedherein complied with specified limits.

Example 5: Proportional Relationship Between Differential Pressure andFlux

To investigate the relationship between pressure differential andvolumetric flow rate, cell culture medium was subjected to filtrationusing autoclaved virus filters at ambient temperature. Media was filledinto a pressure vessel and the virus filters connected to the pressurevessel, which was then pressurized at different levels. The specificflow rate and differential pressures were measured with a balance and apressure transducer and recorded over time (FIG. 3).

Example 6: 10 L Scale-Down Fermentation System Model

A comparison of cell culture media with and without virus filtration wasperformed using a recombinant protein expressing CHO cell fermentationsystem (FIG. 6). The performance with regard to growth rate and yieldswas investigated. Cell culture medium as described in Example 2 wasprepared. One part of the experiment was carried out with only sterilefiltered medium, whereas the other part was carried out with the samemedium and an additional virus filtration using a Sartorius Virosart CPV180 cm². Filtration was carried out at 2-8° C. The fermentationexperiment was carried out in Rushton type agitated 10 L benchtopbioreactors with inline controlled pH, pO2 and temperature. Theparameter setpoints and ranges for the fermentation were as follows:

-   -   pH: 7.05 (6.8-7.3)    -   T: 37.0° C. (35-39° C.)    -   DO: 20% (Air saturation) (10-60%)        Cells were cultivated in batch mode followed by a chemostat        culture using the media with and without additional virus        filtration. Data from the chemostat mode (growth rates and        productivity) were generated from a 4 week continuous cell        culture.

Cell counts were determined by CASY measurement. In chemostat culturethe specific growth rate (p) was calculated by:

μ=D+ln(X _(t1) /X _(t0))/(t ₁ −t ₀)

where D is the dilution rate calculated as ratio of medium feed rate perday and working volume [1/d]. Growth rates were calculated from CASYhomogenized cell counts.

For biochemical analysis, the homogenous suspension was centrifuged with400×g in a Heraeus Multifuge 1 S-R for 10 min and 1.0 mL aliquots wereprepared in Eppendorf tubes and stored at −20° C. Cell free supernatantswere analyzed for the activity of an expressed recombinant protein by achromogenic assay according to standard operating procedures.The volumetric productivity P in this experiment was calculated by:

P [U/(L×d)]=Activity [mU/mL]*dilution rate [d ⁻¹]

The cell-specific productivity qP was calculated by:

qP [mU/(10E06 cells×d)]=P [U/(L×d]/cell count [10E06 cells/mL]

Example 7: 120 L Scale-Down Fermentation System Model

A continuous virus filtration technique performed on a 120 L workingvolume of media prior to its addition to a recombinant proteinexpressing CHO cell fermentation system was investigated with regard toits effect on growth rate and yields (FIG. 6A, FIG. 6B and FIG. 6C). Thestudy compared production processes using three variations of the samecell culture media: a) standard media; b) standard media filtered usingVirosart CPV virus filters; and c) standard media filtered usingMillipore Viresolve NFP virus filters.

During the continuous production process, the two different virusfilters (Virosart CPV Midicap size 2000 cm² and Millipore Viresolve NFPsize 850 cm²) were used alternatively for different time intervals. TheSartorius CPV filter was used from culture day K00-K14, K23-K30 andK39-K63 and the Millipore NFP filter was used from culture day K14-K23and K30-K39.The parameter setpoints and ranges for the fermentation were as follows:

-   -   pH: 7.05 (6.8-7.3)    -   T: 37.0° C. (35-39° C.)    -   DO: 20% (Air saturation) (10-60%)

Sampling and Analysis

Cell counts were determined by CASY cell count and analyzer system. Forbiochemical analysis the homogenous suspension was centrifuged with400×g in a Heraeus Multifuge 1 S-R (Thermo Scientific, USA) for 10 min.Cell free supernatants were analyzed for the activity of an expressedrecombinant protein by a chromogenic assay.In chemostat culture the specific growth rate (p) was calculated by:

μ=D+ln(X _(t1) /X _(t0))/(t ₁ −t ₀)

where D is the dilution rate calculated as ratio of medium feed rate perday and working volume [1/d]. Growth rates are calculated from CASYhomogenized cell counts.The volumetric productivity P in this experiment was calculated by:

P [U/(L*d)]=Activity [mU/mL]*dilution rate [d−1]

The cell-specific productivity qP was calculated by:

qP [mU/(10E06 cells×d)]=P [U/(L*d)]/cell count [10E06 cells/mL]

Example 8: Virusfiltration with ASAHI Planova 15N Virus Filters

Soy hydrolysate containing media (DOMO SE50 MAF #5) were spiked with MMVand placed into a tank connected to a pressurized nitrogen gas supply.The MMV-spiked material was passed through a 10 cm² ASAHI Planova 15Nvirus filter set-up in-line in a dead-end mode at a constant pressure of1100 mbar (set-point). The minimum and maximum values of followingparameters were measured and recorded continuously: Feed pressure; feed,filtrate and ambient temperature and filtrate weight (the change ofwhich was used to calculate the filtrate flow rate). Samples were takendaily for up to 7 days and analyzed for MMV virus titer (FIG. 7).

Example 9: Virusfiltration with ASAHI Planova BioEX Virus Filters

Soy hydrolysate containing media (Run #1 with soy hydrolysate DMV SE50MAF UF #5); Run #2 with soy hydrolysate SDMV SE50 MAF UF #4) were spikedwith MMV and placed into a tank connected to a pressurized nitrogen gassupply. The MMV-spiked material was passed through a 10 cm² ASAHIPlanova BioEX virus filter set-up in-line in a dead-end mode at aconstant pressure of 2000 mbar (set-point). The minimum and maximumvalues of following parameters were measured and recorded continuously:Feed pressure; feed, filtrate and ambient temperature and filtrateweight (the change of which was used to calculate the filtrate flowrate). Samples were taken daily for 5 days and analyzed for MMV virustiter (FIG. 8).

Example 10: Virusfiltration Summary

Cell culture media containing different soy hydrolysates were spikedwith MMV and placed into a tank connected to a pressurized nitrogen gassupply. Different virus filters were used in combination with thedifferent soy hydrolysates as listed in Table 6:

TABLE 6 Combination of virus filters and soy hydrolysates used inspiking experiments Soy hydrolysate Run time Experiment # Filter Lot[days] 1 D 7 5 2 D 6 5 3 I 3 19 4 I 5 17 5 G 7 7 6 G 7 6 7 B 6 14 8 H 311

Filtrations were set-up in a dead-end mode at a constant pressure of 2bar (set-point) for all runs except for the runs Experiments #5 and 6which were performed at a constant pressure of 1.1 bar (set-point). Theminimum and maximum values of following parameters were measured andrecorded continuously: Feed pressure; feed, filtrate and ambienttemperature and filtrate weight (the change of which was used tocalculate the filtrate flow rate). Samples were taken during the runtime of the experiment and analyzed for MMV virus titer. Overall logreductions were calculated from the difference of the total virusinfectivity load in the filtrate and the total virus infectivity loadprior to filtration (FIG. 9).

Example 11: Long Term Filtration with MMV Virus Spike

Cell culture medium as described in Example 2 was spiked with MMV to atiter of 5.0 [log₁₀(TCID₅₀/mL)] and subjected to a long term filtrationof 30 days over a 20 nm pore-size viral filter (Sartorius Visrosart CPV5 cm²). Filtration was carried out with a set-up comparable to Example 9and Example 10, but with constant pressure of 1.1 bar (specified range:0.8 bar to 1.2 bar) and with regular pressure and flow interruptions tochallenge the viral filter. Flow rates in the course of the experimentwere recorded and maintained above 4 L/(m²×hr) (FIG. 10).

20 samples of the filtrate were taken (up to 5 times per week) and theMMV virus titer and load determined. No virus break-through was observedin any of the 20 fractions assayed. Virus loads ranged from <0.9[log₁₀(TCID₅₀)] to <2.8 [log₁₀(TCID₅₀)] depending on fraction volume.The total virus load in the filtrates was <3.0 [log₁₀(TCID₅₀)]which—when subtracted from the initial virus load of the spiked material(i.e. 8.5 [log₁₀(TCID₅₀)])—results in an overall virus reduction factorof >5.5 log₁₀. This was seen to be effective and complete (FIG. 11).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1.-14. (canceled)
 15. An apparatus comprising a bioreactor and a virusfilter, wherein the virus filter is inline of the medium feed line ofthe bioreactor, wherein the virus filter has an effective pore size ofmaximum 75 nm, and wherein the virus filter is for removing a viralcontaminant from a preparation, said preparation being a cell culturemedium or at least one component of a cell culture medium.
 16. Theapparatus of claim 15, wherein the bioreactor is a chemostat reactor, aperfusion reactor, or a fed batch reactor.
 17. The apparatus of claim15, wherein the filter comprises two or more filters arranged in series,in parallel, or both.
 18. The apparatus of claim 17, wherein the filtercomprises two filters arranged in parallel in a system of tubescomprising a Y-shaped junction and wherein each filter is in fluidcommunication with a branch of the Y-shaped junction and a preparationsupply source.
 19. The apparatus of claim 15, wherein the virus filteris autoclavable.
 20. The apparatus of claim 15, wherein the cell culturemedium is a cell culture medium comprising a soy hydrolysate or a cellculture medium comprising animal derived components.
 21. A cell culturemedium prepared by a method comprising a) subjecting a preparation tofiltration for at least about 24 hours through a virus filter having aneffective pore size of maximum 75 nm, wherein the preparation is a cellculture medium or at least one component of a cell culture medium, andwherein the filtration operates at a volumetric capacity of at leastabout 2000 L/m².
 22. The cell culture medium of claim 21, wherein thefiltration operates at a volumetric capacity of at least about 3000L/m², or at least about 5000 L/m².
 23. The cell culture medium of claim21, wherein the preparation is subjected to filtration for at leastabout 48 hours to about 7 months, or for at least about 72 hours toabout 3 months, or for at least about 10 days to about 2 months.
 24. Thecell culture medium of claim 21, further comprising b) feeding thefiltrate to a cell culture medium or to at least one component of a cellculture medium.
 25. The cell culture medium of claim 21, wherein thefiltration is a continuous filtration.
 26. The cell culture medium ofclaim 21, wherein the filtration is performed at a temperature of fromabout 2° C. to about 60° C., or from about 10° C. to about 40° C., orfrom about 15° C. to about 37° C.
 27. The cell culture medium of claim21, wherein the virus filter achieves at least a 1 Log₁₀ reduction value(LRV) for a viral contaminant, or at least a 4 Log₁₀ reduction value(LRV) for a viral contaminant, or at least a 6 Log₁₀ reduction value(LRV) for a viral contaminant.
 28. The cell culture medium of claim 27,wherein the viral contaminant is a member of the viral family ofOrthomyxoviridae, Arenaviridae, Paramyxoviridae, Rhabdoviridae,Coronaviridae, Flaviviridae, Picornaviridae, Togaviridae, Arteriviridae,RetParvoviridae, Bunyaviridae, Caliciviridae, Retroviridae, Reoviridae,Circoviridae, Adenoviridae, Poxviridae, Herpesviridae, Iridoviridae orReoviridae.
 29. The cell culture medium of claim 21, wherein thefiltration is performed using two or more filters arranged in series, inparallel, or both.
 30. The cell culture medium of claim 29, wherein thefiltration is performed using two filters arranged in parallel in asystem of tubes comprising a Y-shaped junction and wherein each filteris in fluid communication with a branch of the Y-shaped junction and apreparation supply source.
 31. The cell culture medium of claim 21,wherein the filtration is performed at a pressure ranging from about 100mbar to about 4000 mbar, or from about 100 mbar to about 3500 mbar, orfrom about 1000 mbar to about 3000 mbar, or from about 1000 mbar toabout 2000 mbar.
 32. The cell culture medium of claim 21, wherein thevirus filter is autoclavable.
 33. The cell culture medium of claim 21,wherein the cell culture medium comprises soy hydrolysate.
 34. A methodcomprising utilizing the cell culture medium of claim 21 for preparing acell culture, a pharmaceutical preparation, a diagnostic preparation, acosmetic preparation or a food preparation.